DNA adduct formation by o-phenylphenol metabolite in vivo and in vitro.

[U-14C]o-Phenylphenol (OPP) was found to bind covalently to calf thymus DNA during a 60 min incubation in the presence of microsomes, but not in their absence, indicating that metabolic conversion of the parent compound, OPP, to an activated form is essential. Postlabeling analysis with bladder DNA of rats fed a diet containing 2% OPP for 13 weeks revealed one major adduct on TLC. In an in vitro postlabeling experiment with calf thymus DNA, both of the major metabolites of OPP, phenylhydroquinone (PHQ) and phenylbenzoquinone (PBQ), formed adducts, but no adducts were observed with OPP. The chemical structure responsible for adduct formation is thought to be the PHQ semiquinone radical intermediate formed during interconversion between PHQ and PBQ. When the oligonucleotides, pd(A)12-18, pd(C)12-18, pd(G)12-18 and pd(T)12-18, were used in vitro, only pd(G)12-18 gave TLC-detectable adducts on treatment with PHQ and PBQ. The covalent binding appears to be rather specific to guanine residues. These results suggest that covalent binding of the OPP metabolite is one of the underlying events in OPP-induced carcinogenesis in rats.     

DNA adduct formation of aristolochic acid I and II in vitro and in vivo

Aristolochic acid I (AA I) and aristolochic acid II (AA II),the two main ingredients of the carcinogenic plant extract aristolochicacid (AA), are metabolized to reactive intermediates which bindcovalently to DNA in vitro and in vivo. DNA adduct formationwas analysed by the ³²P-postlabelling assay. In in vitro incubationswith rat liver 9000 g supernatant (S9) and calf thymus DNA (CT-DNA),AA I showed an identical pattern of DNA adducts on thin-layerchromatograms under aerobic and anaerobic conditions, whereasAA II gave rise to DNA adduct formation only anaerobically.The anaerobically obtained DNA adduct pattern by AA II in vitrowas similar to the AA I adduct patterns. Aristolactams I andII, the metabolites of AA I and AA II formed under anaerobicconditions, did not form DNA adducts in the presence of S9 mixand CT-DNA. Incubations with xanthine oxidase, known to enzymaticallyreduce aromatic nitro groups, also activated AA I and AA IIto reactive intermediates, producing almost identical adductpatterns as obtained by S9 mix-mediated metabolism. Activationof AA I by S9 mix in the presence of poly(dG) resulted in theformation of two adducts, one of which was shown to be chromatographicallyindistinguishable from an adduct obtained by reaction with CT-DNA.For the in vivo studies AA I and AA II were administered orallyto male Wistar rats, and DNA from liver, brain, oesophagus,stomach lining, forestomach lining, kidney and bladder was analysedfor DNA adducts by ³²P-postlabelling. The adduct patterns inDNA from forestomach and kidney—target tissues of AA—andDNA from non-target tissues like stomach lining and liver weresimilar to the patterns obtained from the in vitro incubations.In the bladder (also a target tissue) only AA II gave rise toDNA adduct formation. These findings suggest that DNA adductformation by AA I and AA II does not directly correlate withthe initiation of the carcinogenic process and subsequent tumourformation in target tissues in the rat.     

Identification and characterization of the major DNA adduct formed chemically and in vitro from the environmental genotoxin 3-nitrofluoranthene

The genotoxic environmental pollutant 3-nitrofluoranthene (3-NFA)was reduced chemically and allowed to react with calf thymusDNA, yielding one major adduct which was determined to be N-deoxyguanosin-8-yl)-3-amino-fluoran-thenebased on Fast Atom Bombardment Mass Spectrometry (FAB-MS), protonnuclear magnetic resonance, ultra-violet-visible wavelengthlight spectroscopy (UV-VIS), and fluorescence data. Extensivecharacterization of the isolated adduct by tandem mass spectrometry(MS/MS) was necessary to demonstrate definitively that the adductisolated was the dG: C8 adduct, and not the isomeric dG: N2adduct. The extent of modification of the initial calf thymusDNA by chemically reduced 3-NFA was 0.12% (1.2 adducts/10P nucleosides),which was sufficient to allow several hundred micrograms ofthe adduct to be isolated and purified. The chemically synthesizedadduct was utilized as a reference standard for comparison tothe major adduct isolated from xanthine-oxidase-catalyzed reductionof 3-NFA in vitro. The yield from the in vitro biological systemwas 2.4 adducts/10⁵ nucleosides; the adduct isolated possessedthe same mass spectrometric. UV-VIS, and fluorescence characteristicsas the purified standard, and co-eluted with the standard onHPLC. No evidence for other adducts was found, either in vitroor in the chemical synthesis, based on FAB-MS examination ofwhole extracts of the reaction mixture for the presence of ionsrelated to other possible adducts. Therefore, if minor adductswere present they were formed in substantially lesser amountsthan N-(deoxyguanosin-8-yl)-3-aminoflouranthene.     

32P-Postlabelling of DNA adducts formed by allyl glycidyl ether in vitro and in vivo

32P-Postlabelling analysis of allyl glycidyl ether-treated DNA after adduct enrichment on anion-exchange cartridges revealed two major and one minor DNA adducts. The major adducts were shown to originate from alkylation at N-7-guanine and N-1-adenine, respectively, while the minor adduct was at N-3-cytosine. In addition, rearrangement products of the 1-adenine and 3-cytosine adducts to N6-adenine and 3-uracil were indicated. The relative amounts of adenine, cytosine and uracil products appeared to be dependent upon conditions (in particular pH) during sample processing and analysis. When nuclease P1 was used for adduct enrichment the adenine, cytosine and uracil adducts, but not the 7-guanine adduct, were detected. The labelling efficiency of the 7- guanine adduct standard was 40-45%. Total recovery of this adduct from allyl glycidyl ether-modified DNA was 9-12%. The labelling efficiency of the 1-adenine adduct standard was 78-82%. Total recovery of this adduct from DNA was approximately 20% when using anion-exchange chromatography for adduct enrichment and 30-34% when using nuclease P1. Preliminary analysis of DNA from mice treated with allyl glycidyl ether indicated 57 times higher level of the 7-guanine adduct, per unit dose, in skin DNA (120 per 10(8) normal nucleotides) after topical application when compared to liver DNA after i.p. administration. The 1- adenine adduct could not be quantified in liver DNA (due to an interfering background product present in untreated animals) and the level of the 3-cytosine adduct was below the detection limit of the method. After topical application the level of the 1 adenine adduct in skin DNA was approximately 30 per 10(8), using either column or nuclease P1 enrichment. The 3-cytosine adduct was detected in skin, but was not quantified.      

Androgen manipulation alters oxidative DNA adduct levels in androgen-sensitive prostate cancer cells grown in vitro and in vivo

Intracellular reactive oxygen species (ROS) may cause oxidative DNA damage, resulting in the formation of adducts such as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and the cyclic pyrimidopurinone N-1, N(2) malondialdehyde-2'-deoxyguanosine (M(1)dG). These adducts have been associated with carcinogenesis, genomic instability and clonal evolution. We tested two hypotheses in human prostate cancer cells grown in vitro and in a xenograft model: (1) treatment of androgen-sensitive cells with DHT increases levels of oxidative DNA adduct levels; (2) flutamide, a competitive androgen receptor antagonist, prevents DHT-induced changes. Levels of M(1)dG and 8-oxo-dG adducts were determined by immunoslot blot and liquid chromatography-tandem mass spectrometry. M(1)dG and 8-oxo-dG levels were significantly higher than control levels in LNCaP cells exposed to supra-physiological concentrations (25-100 nM) of DHT (both P<0.05 by ANOVA). Flutamide pre-treatment completely prevented this increase. In the xenograft model, tumour levels of M(1)dG were decreased by 46% (P=0.001 by Mann-Whitney Test) in flutamide-treated animals compared to controls. The changes demonstrated suggest that oxidative DNA adducts may serve as biomarkers of the efficacy of androgen manipulation in chemoprevention trials.     

Co-chromatography of a tamoxifen epoxide-deoxyguanylic acid adduct with a major DNA adduct formed in the livers of tamoxifen-treated rats

Tamoxifen is a potent liver carcinogen in rats and has beenshown to form covalent DNA adducts in the livers of severalspecies of rodent. We have shown previously by ³²P-postlabelling(Carcinogenesis, 13, 2197–2203) that >85% of the totaladducts detected and resolved by multi-directional TLC migrateas a single spot. In the present study, this material was furtheranalysed by reverse-phase HPLC and resolved into two approximatelyequal components. Tamoxifen 1,2-epoxide, a postulated metaboliteof tamoxifen, was reacted with DNA and polydeoxyribonucleotidesand the products analysed. ³²P-Postlabelling revealed threemajor adduct spots on TLC which comigrated with the three majoradduct spots seen with DNA from livers of tamoxifen-treatedrats. Moreover, the major epoxide adduct, which contained guanineas the modified base, eluted on HPLC as a single major peakcoincident with one of the major peaks derived from the liverDNA of tamoxifen-treated rats. These results demonstrate that{small tilde}40% of the tamoxifen-DNA adducts formed in vivoare chromatographically indistinguishable with the major productof the reaction of tamoxifen epoxide with guanine residues inDNA and provide important clues to the mechanism of activationof tamoxifen to a genotoxic carcinogen.     

Identification of the major serum albumin adduct formed by 4-aminobiphenyl in vivo in rats

Serum albumin was isolated from rats at 27 h after administration of the carcinogen [2,2'-3H]-4-aminobiphenyl. Pronase digestion of the purified albumin yielded a mixture of radiolabeled materials which was resolved into 5 major components by reverse-phase liquid chromatography. From detailed UV, 1H-NMR, and mass spectral analyses, four of these were determined to be 4-aminobiphenyl, 4'-hydroxy-4-acetylaminobiphenyl, and two other metabolites, all of which are presumed to be non-covalently associated with the serum albumin. The fifth component, however, resulted from covalent bond formation and was identified as a tetrapeptide containing 3-tryptophanyl-4-acetylaminobiphenyl, the amino acid sequence of which was H2N-ala-trp-ala-val. Since rat serum albumin contains only a single tryptophan residue in a hydrophobic drug binding site, its high selectivity for carcinogen binding suggests a unique role for this protein in the detoxification and/or transport of ultimate carcinogenic metabolites.     

DNA adduct formation and unscheduled DNA synthesis in rat esophagus in vivo after treatment with N-methyl-N-nitrosourea

An in vivo method for assessment of DNA adduct formation and unscheduled DNA synthesis (UDS) in the esophagus of rats was devised. Small ventral incisions were made in the neck and upper abdomen regions of 6 week old F344 rats and ligation of the esophagus with thread at the two extreme ends performed to make an esophageal pouch. For the DNA adduct formation study, a solution (0.5 ml) containing various concentrations of N-[3H]methyl-N-nitrosourea ([3H]MNU) was injected into the pouch. DNA binding levels were calculated from radioactivity of the isolated DNA and dose-dependent DNA adduct formation could be detected 2 h after the treatment with MNU. By HPLC analysis, both 7-methylguanine (7-mGua) and O6-methylguanine (O6-mGua) adducts were identified in the esophageal DNA, the ratio of 7-mGua/O6-mGua being 5.7-12:1. For UDS measurement, a solution containing MNU plus [3H]thymidine (200 microCi/ml) was similarly injected into the pouch. UDS was dose-dependently demonstrated as silver grains over the nuclei of the epithelial cells by autoradiography. The results thus showed that MNU, when injected into the esophageal lumen, can penetrate the surface mucosa, react with the epithelial cell DNA and induce DNA adduct formation and DNA repair synthesis dose-dependently.     

Identification of the DNA adduct formed by metabolism of 1,8-dinitropyrene in Salmonella typhimurium

The incubation of [³H]1,8-dinitropyrene with Salmonella typhimuriumTA98NR followed by isolation of the DNA from these cells, hydrolysisof the DNA to nucleosides, butanol extraction of the hydrolysateand purification by reversed-phase liquid chromatography affordeda single product. Calf thymus DNA, after treatment with N-hydroxyl-1-amino-8-nitropyrene,was hydrolyzed, extracted and purified in a similar fashionto give a single compound which was shown to be the deoxyguanosinederivative 1-N-(2'-deoxyguanosin-8-yl)-amino-8-nitropyrene bya combination of proton n.m.r. and u.v.-vis. spectroscopy andfast atom bombardment mass spectrometry. The DNA adducts formedin vivo and in vitro exhibited identical chromatographic andchemical behavior. Under acidic or basic conditions the in vivoand in vitro adducts were converted to identical products. Reductionof the adduct gave a new, highly fluorescent product that hada fluorescent emission spectrum identical to that of 1,8-diaminopyrene.     

Correlation between DNA or protein adducts and benzo[a]pyrene diol epoxide I-triglyceride adduct detected in vitro and in vivo

In this study, we demonstrated the in vitro and in vivo formation of carcinogen-lipid adduct and its correlation with DNA or protein adducts. The lipids from serum or hepatocyte membranes of Sprague-Dawley rats, human serum and standard major lipids were in vitro reacted with benzo[a]pyrene (B[a]P) and B[a]P metabolites. 7, 8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE-I), an ultimate carcinogenic form of B[a]P, was covalently bound to triglyceride (TG). BPDE-I-TG adducts isolated by thin-layer chromatography (TLC) were further detected by high-performance liquid chromatography. TGs, including triolein, tripalmitin and tristearin, showed positive reactions with BPDE-I. However, cholesterol, phospholipids (phosphatidylcholine, phosphatidyl-ethanolamine, phosphatidyl-inositol and sphingomyelin) and non-esterified fatty acids (palmitic acid, oleic acid, linoleic acid and stearic acid) did not react with BPDE-I. In addition, other B[a]P metabolites (B[a]P-phenols and -diols) did not react with TG. TG appeared to be the most reactive lipid yet studied with respect to its ability to form an adduct with BPDE-I. There was a clear-cut dose-related formation of [1,3-(3)H]BPDE-I-lipid adducts in vitro between TG and [1,3-(3)H]BPDE-I. In an animal study, BPDE-I-TG was also formed in the serum of rats orally treated with B[a]P (25 mg/rat). Also, obvious correlations between [(3)H]B[a]P related-biomolecule adducts (DNA or protein) or lipid damage and the BPDE-I-TG adducts were obtained in various tissues of mice i.p. treated with [(3)H]B[a]P. These data suggest that TG can form an adduct with BPDE-I, as do other macromolecules (DNA, RNA and protein). Therefore, a carcinogen-lipid adduct would be a useful biomarker for chemical carcinogenesis research and cancer risk assessment.     

Isolation and characterization of the major serum albumin adduct formed by aflatoxin B1 in vivo in rats

Aflatoxin B₁ (AFB₁) was shown to react primarily with one ormore lysine residues in serum albumin (SA), accounting for morethan half of the total binding to this protein. The radioactivityassociated with SA following administration of [U-¹⁴C]AFB₁ torats was cleared with a half-life of 2.5 days, which is notsignificantly different from the half-life of unmodified albuminin the normal rat. The product isolated from a Pronase digestof in vivo-modified SA was identical by chromatographic retentiontime and u.v. and mass spectroscopy to the synthetic productobtained by the acylasecatalyzed deacetylation of the reactionproduct of N-acetyl-L-lysine with 8,9-dihydro-8,9-dibromo-AFB₁.The latter was characterized by u.v., fluorescence, 500 MHz¹H-n.m.r. and fast atom bombardment mass spectrometry. The spectraldata strongly support a structure in which the terminal dihydrofuranring of AFB₁ has been converted to a pyrrolinone ring. It isproposed that the initial adduct is formed by condensation ofthe dialdehyde tautomer of 8,9-dihydro-8,9-di-hydroxy-AFB₁,with the -amino group of lysine, to form a Schiff base, andthat the Schiff base undergoes an Amadori rearrangement to an-amino ketone. The pyrrolinone ring is formed by condensationof the amino group with the remaining aldehyde to yield thefinal product. The purified product was relatively stable butwas shown to decompose significantly under the conditions usedto isolate it from modified SA.     

In vivo genotoxicity and DNA adduct levels in the liver of rats treated with safrole

The induction of chromosome aberrations, sister chromatid exchanges (SCEs), and the formation of DNA adducts was studied in hepatocytes of F344 rats exposed in vivo to safrole. Hepatocytes were isolated 24 h after a single dose of safrole or five repeated doses (once a day) by gastric intubation and allowed to proliferate in Williams' medium E supplemented with epidermal growth factor. Cells were fixed after 48 h in culture. Safrole-DNA adducts were detected by a nuclease P1-enhanced 32P-post-labeling assay in isolated hepatocytes from the rats. While a single dose was not sufficient to induce detectable levels of chromosome aberrations at the time of assay, five repeated doses induced these changes with a maximum frequency of 13.4%, compared with the control value of 1.8%. Both a single dose and five repeated doses induced significant SCEs, to a maximum frequency of 0.81 SCEs per chromosome, while the control value was 0.59 SCEs per chromosome. Two major and two minor DNA adducts were detected after treatment with either a single dose or five repeated doses. The maximum amount of total DNA adducts was 89.8 DNA adducts/10(7) nucleotides. These results show that safrole is a genotoxic carcinogen in the rat liver in vivo and suggest that the cytogenetic effects of this compound may result from covalent DNA modification in the rat liver. This in vivo cytogenetic assay should provide a useful means of evaluation of the genotoxicity of hepatocarcinogens.      

The cytotoxic action of adriamycin and cyclophosphamide on tumor cells in vitro and in vivo

The ability of Adriamycin (ADR) to kill tumor cells were studied using two transplantable murine tumors adapted for growth in tissue culture. Cells were treated either in vivo as subcutaneous (SC) tumors or lung colonies up to 2 mm in diameter or in vitro. ADR in contact with cells in vitro for one hour readily killed cells from both CBSAF and WHFIB tumors. Cells plated from tumors were more resistant in vitro than cells kept in culture. The D₀'s were 0.17 and 0.1 gm/ml, respectively. The resistant tail in WHFIB at 10^?2 survival was not inherited by surviving cells. In log phase the D was reduced. However, in vivo an IP or IV dose of 18 μ/ml ADR to mice bearing CBSAF tumors produced a surviving fraction as high as 0.64 for SC tumors and 0.3 for lung colonies.Hypoxic tumor cells in vitro are more resistant than aerobic ones; there may also be a tumor size effect because lung colony cells treated in vitro has a sensitivity intermediate between SC tumors and cultured cells; but even both factors together cannot explain the lack of effect in vivo relative to in vitro. Preliminary studies with cyclophosphamide suggested that it, in contrast to ADR, was highly effective in killing tumor cells both in SC tumors and in lung colonies.     

Cytotoxicity,DNA cross-linking,and DNA single-strand breaks induced by cyclophosphamide in a rat leukemia in vivo

Summary A study of cyclophosphamide (CP)-induced DNA damage and repair occurring in vivo was conducted in the brown Norway rat myelocytic leukemia (BNML) model. DNA single-strand breaks (SSB), DNA-DNA interstrand cross-links (DIC), DNA-protein cross-links (DPC), and DNA double-strand breaks (DSB) were measured by alkaline and neutral elution. After i. p. injection of 50 mg/kg CP, DIC were detectable at 1 h and peaked at 8 h. DPC were detectable at 2 h and peaked at 6 h. Both DIC and DPC persisted at a relatively high level until 28 h. Dose-response curves for both DIC and DPC were determined at 4 h after CP injection over the dose range of 25–150 mg/kg. These doses ranged from the minimally effective dose to doses curative for rats bearing this leukemia (1- to 9-log kill of leukemia cells). No SSB or DSB was observed at 4 h after CP injection over the dose range of 15–250 mg/kg, but a low level of SSB was observed at 18–28 h after CP treatment. These data suggest that the cytotoxic effect of CP in vivo is mediated mostly by DIC and DPC. SSB appearing late after CP injection in vivo may be a reflection of repair of DIC and DPC and an indication of the optimal timing for administration of DNA-repair inhibitors. This observation is of interest since our earlier work demonstrated that hydroxyurea can potentiate the therapeutic benefit of CP in this model when it is given over the 4-day period immediately after CP treatment.Supported by NIH grant RO1 CA455329     

Comparison of the DNA adducts formed by tamoxifen and 4-hydroxytamoxifen in vivo

Tamoxifen is a liver carcinogen in rats and has been associated with an increased risk of endometrial cancer in women. Recent reports of DNA adducts in leukocyte and endometrial samples from women treated with tamoxifen suggest that it may be genotoxic to humans. One of the proposed pathways for the metabolic activation of tamoxifen involves oxidation to 4-hydroxytamoxifen, which may be further oxidized to an electrophilic quinone methide. In the present study, we compared the extent of DNA adduct formation in female Sprague-Dawley rats treated by gavage with seven daily doses of 54 micromol/kg tamoxifen or 4-hydroxytamoxifen and killed 24 h after the last dose. Liver weights and microsomal rates of ethoxyresorufin O-deethylation, 4-dimethylaminopyrine N-demethylation and p-nitrophenol oxidation were not altered by tamoxifen or 4-hydroxytamoxifen treatment. Uterine weights were decreased significantly and uterine peroxidase activity was decreased marginally in treated as compared with control rats. DNA adducts were assayed by 32P-post-labeling in combination with HPLC. Two major DNA adducts were detected in liver DNA from rats administered tamoxifen. These adducts had retention times comparable with those obtained from in vitro reactions of alpha-acetoxytamoxifen and 4-hydroxytamoxifen quinone methide with DNA. Hepatic DNA adduct levels in rats administered 4-hydroxytamoxifen did not differ from those observed in control rats. Likewise, adduct levels in uterus DNA from rats treated with tamoxifen or 4-hydroxytamoxifen were not different from those detected in control rats. These data suggest that a metabolic pathway involving 4-hydroxytamoxifen is not a major pathway in the activation of tamoxifen to a DNA-binding derivative in Sprague-Dawley rats.     

Factors modulating the formation of DNA adduct by aflatoxin B1 in vitro

Forty-two compounds belonging to various chemical groups havebeen tested for their ability to suppress formation of aflatoxinB₁-DNA adduct mediated by microsome in vitro. While many ofthese compounds have either marginal or no modulating effect,some have been identified as effective inhibitors. The stronginhibition of DNA adduct formation by retinoids (retinol, retinal,retinoic acid and retinyl acetate), riboflavin, riboflavin 5'-phosphate,flavin adenine dinudeotide, Cu²⁺, 7,8-benzoflavone, disulfiram,butylated hydroxyanisole, butylated hydroxytoluene and phenothiazinesuggests that these agents may have potential anticardnogenicactivity against aflatoxin B₁.     

32P-postlabeling analysis of peroxisome proliferator-DNA adduct formation in rat liver in vivo and hepatocytes in vitro

Hepatocarcinogenic peroxisome proliferators, clofibrate, ciprofibrate, Wy-14643 or di(2-ethylhexyl)phthalate, were administered once daily by gavage to groups of three male F344 rats for 3 days and the rats were killed 2 h after the last dose. The DNA isolated from the livers was analyzed for possible carcinogen-DNA adducts, by a most sensitive 32P-postlabeling technique which can detect one adduct in 10(10) nucleotides. No adducts were detected by this assay in the DNA isolated from the livers of rats treated with any of the peroxisome proliferators. Adducts were also not found in the DNA of hepatocytes exposed in vitro to these peroxisome proliferators for 4 h in primary suspension cultures. Failure to detect peroxisome proliferator DNA adducts in hepatocytes under in vivo and in vitro conditions supports the contention that formation of a peroxisome proliferator-DNA adduct is not an essential step in the carcinogenesis by this novel class of carcinogens.     

Combination of N,N',N"-triethylenethiophosphoramide and cyclophosphamide in vitro and in vivo

In vitro and in vivo studies with the drug combination thioTEPA and cyclophosphamide (CPA) were carried out using the MCF-7 human breast carcinoma cell line and the EMT6 mouse mammary carcinoma cell line. In vitro, survival curves were essentially linear. The EMT6 cell line was less sensitive to thioTEPA than the MCF-7 cell line, with concentrations which reduce cell survival to 10% of 440 and 140 microM, respectively. The response of both cell lines to 4-hydroperoxycyclophosphamide was similar. Simultaneous and immediate sequential treatments with these drugs produced supraadditive cell killing of both cell lines, although the magnitude of the supraadditivity was greater in the MCF-7 cell line than in the EMT6 cell line. Both of these drugs appeared to be as effective as thiol-depleting agents as is diethyl maleate. By DNA alkaline elution, there was a pattern of increasing DNA cross-linking similar to the increasing levels of cytotoxicity of this drug combination with increasing thioTEPA concentrations. In the EMT6 tumor in vivo, the maximally tolerated combination therapy (5 mg/kg x 6 thioTEPA and 100 mg/kg x 3 CPA) produced about 25 days of tumor growth delay which was not significantly different than expected for additivity of the individual drugs. The survival of EMT6 tumor cells after treatment of the animals with various single doses of thioTEPA and CPA was assayed. Tumor cell killing by thioTEPA produced a very steep, linear survival curve through 5 logs. The tumor cell survival curve for CPA out to 500 mg/kg gave linear tumor cell kill through almost 4 logs. In all cases, the combination treatment tumor cell survivals fell well within the envelope of additivity. Both of these drugs are somewhat less toxic toward bone marrow cells by the granulocyte-macrophage colony-forming unit in vitro assay method than to tumor cells. The combination treatments were subadditive or additive in bone marrow granulocyte-macrophage colony-forming unit killing. When bone marrow is the dose-limiting tissue, there is a therapeutic advantage to the use of this drug combination.     

Pharmacodynamics of cisplatin in human head and neck cancer: correlation between platinum content, DNA adduct levels and drug sensitivity in vitro and in vivo.

Total platinum contents and cisplatin-DNA adduct levels were determined in vivo in xenografted tumour tissues in mice and in vitro in cultured tumour cells of head and neck squamous cell carcinoma (HNSCC), and correlated with sensitivity to cisplatin. In vivo, a panel of five HNSCC tumour lines growing as xenografts in nude mice was used. In vitro, the panel consisted of five HNSCC cell lines, of which four had an in vivo equivalent. Sensitivity to cisplatin varied three- to sevenfold among cell lines and tumours respectively. However, the ranking of the sensitivities of the tumour lines (in vivo), also after reinjection of the cultured tumour cells, did not coincide with that of the corresponding cell lines, which showed that cell culture systems are not representative for the in vivo situation. Both in vitro and in vivo, however, significant correlations were found between total platinum levels, measured by atomic absorption spectrophotometry (AAS), and tumour response to cisplatin therapy at all time points tested. The levels of the two major cisplatin-DNA adduct types were determined by a recently developed and improved 32P post-labelling assay at various time points after cisplatin treatment. Evidence is presented that the platinum-AG adduct, in which platinum is bound to guanine and an adjacent adenine, may be the cytotoxic lesion because a significant correlation was found between the platinum-AG levels and the sensitivities in our panel of HNSCC, in vitro as well as in vivo. This correlation with the platinum-AG levels was established at 1 h (in vitro) and 3 h (in vivo) after the start of the cisplatin treatment, which emphasizes the importance of early sampling.